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The time course of osmotic regulation in the freshwater shrimp Macrobrachium olfersii (Wiegmann) (Decapoda, Palaemonidae)
245
.I. Exp. Mar. BioL Ecol., 1987, Vol. 107, pp. 245-251 Eisevier
JEM 00858
The time course of osmotic regulation in the freshwater shrimp Macrobrachium olfrsii (Wiegmann) (Decapoda, Palaemonidae) John C. McNamara ~e~~rtarne~to de ~~~o~o~.a,~~ti~to de Bioci&cias, U~iveTsidade de &To Pa&,
&lo Pa&o. Brazil
(Received 10 September 1986; revision received 8 January 1987; accepted 15 January 1987) Abstract: Alterations in hemolymph osmotic concentration (HOC) in the decapod shrimp Macrobrachium olfersii (Wiegmann) were examined as a function of salinity (0, 7, 14,21,28 and 35 S) and exposure time (1,3,6,12 and 24 h). This freshwater palaemonid maintains a high HOC in river water (362.9 + 7.8 mOsm) which is virtually unaffected by salinity variation up to 14 S or exposure time. However, in 21 and 28 S, HOC increases rapidly until 6 h, remaining stable to 12 h, then increasing to 24 h; in 35 S, HOC increases continually with time. M. olfersii hyperregnlates its HOC in salinities up to isosmotic (14 S), exhibiting various degrees of HOC response (strong hyporegnlation to isosmoticity) in higher salinities, as a function of time. Hemoiymph isosmotic points increase little during the first 6 h of exposure, rising rapidly after 12 and 24 h. Given its high HOC in freshwater, tolerance of high salinities and the dependence of its larval stages on saline water, M. o&r&is considered to be a recent, ~thou~ efficient, colonizer of the freshwater biotope. Key words: Macrobrachium; Osmotic regulation; Freshwater shrimp; Salinity exposure
Freshwater shrimps, particularly those of the genus Macrobrachium, are apparently still in the process of penetrating the freshwater biotope (Ortmann, 1902; Hedgpeth, 1949, 1957). Such decapod crustaceans exhibit numerous biological characteristics indicative of the recency of this invasion, e.g., high hemol~ph osmo-ionic concentrations, tolerance of high salinities, marked dependence on saline water for larval development with many larval stages, and migratory behavior. Evidently, the development of an efficient osmoregulatory capabiity is of fundamental importance to the colonization of the freshwater habitat by such shrimps since these maintain high ionic concentrations in the body fluids against a strong gradient, implying considerable energetic expense (Moreira et al., 1983). Investigation of osmo-ionic equilibrium in the hemolymph permits evaluation of the establishment, maintenance and efficiency of the osmotic response which, together with the biological characteristics of the species, should provide a measure of its capability to invade the freshwater biotope Correspondence address: John C. McNamara, Departamento de Fisiologia, Instituto de Cikrcias Biomedicas, CP 4365, Universidade de Sfio Paulo, OlOOOS&J Paulo, Brazil. 0022-~81/87/$03.50 0 1987 Elsevier Science Pnbhshers B.V. (Biomedic~ Division)
246
J. C. McNAMARA
and the adaptive mechanisms concerned (see Beadle, 1943; Shaw, 1961). It is also interesting to evaluate the extent to which other major energy consuming processes such as respiratory meta~lism and excretion, for example, are affected by hemol~ph osmotic response. Some aspects of both osmoregulatory phenomena (see Armstrong et al., 198 1; Castille & Lawrence, 198 1; Moreira et al., 1983; Read, 1984 for references) and respiratory metabolism (see Moreira et&, 1983; McNamara et al., 1986 for references) have been examined in Macrobracbium, and in considerable detail in other brackish water and marine palaemo~d shrimps (see Knowlton & Kirby, 1984; Kirkpatrick & Jones, 1985 for references). Relatively little is known about the regulation of hemolymph osmotic concentration in freshwater shrimps (see Moreira et al., 1983; Read, 1984) and information is especially lacking on the establishment of osmotic response patterns to acute sahnity exposure. Thus, in the present study, I have investigate the time course of hemol~ph osmotic response to salinity variation in a small palaemonid shrimp, M. oifersii (Wiegmarm), in an attempt to evaluate the degree of osmotic adaptation to freshwater in this decapod typical of the coastal fluvial system of southern Brazil. MATERIALS AND METHODS
Specimens of the freshwater shrimp M. o&xv?, 5 to 7 cm total length, were collected from the marginal vegetation of the GuaecL River (23 ‘49’S : 45’ 27’W) on the northern coast of the State of Sao Paulo, Brazil. In the laboratory, the shrimps were rn~nt~~ unfed, in plastic tanks containing 15 1 of aerated river water, at room temperature (x 25 ‘C), for 1 or 2 days prior to experimentation. To evaluate the effect of salinity and exposure time on hemolymph osmotic responses, groups of 15 to 20 intermolt shrimps were exposed for 1, 3,6, 12 or 24 h to media of 0, 7, 14, 21, 28 or 35 S* in a constant temperature chamber fmished with a 12-h light : 12-h dark photoperiod, at 20 “C. All salinities were prepared from Guaecit River water (< 1 S) and Sao Sebastigo Channel sea water (35 S), To obtain hemolymph samples, after each exposure period, the pericardial region of each shrimp was punctured with a Number 25-8 needle and 70 to 100 ~1 of lluid drawn into a 1 ml plastic syringe. The entire volume was then transferred to a 1.5 ml Teflon flask and a 50 ~1 subsample taken with an automatic pipette. The osmotic concentration of this subsample was immediately determined using a Knauer semi-micro osmometer. All results are presented as mOsm/kg water (mean _+SEM).The data were evaluated using the Student-Neck-Keuls procedure and a two-way analysis of variance according to Zar (1974).
* Practicalsalinity:definition (UNESCO TechnicaI Papers in Marine Science, Number 39, Vol. 3, p. 11).
OSMOTICREGULATIONIN ~ACROB~CHIU~
247
RESULTS Data demons~ating the effect of exposure time and salinity on the hemol~ph osmotic ~oncen~ation (HOC) ofM. orfrsii are presented in Table I. In river water (0 S), the normal external medium, HOC remained stable at 362.9 4 7.8 mOsm/kg (n = 55) (mean of all river water exposure times) and was independent of exposure time. In 7 S, no significant differences were recorded among the diBerent exposure times although there was a tendency for HOC to increase with time. In 14 S, the only si~c~t increase was recorded between 0- and 12-h exposure. In 21 S, HOC increased signiticantly from 0 to 1 h and again from 1 to 6 h. There was no difference between the values for 6 and 12 h; HOC increased again significantly from 12to 24 h. A very similar pattern was observed in 28 S, HOC increasing significantly from 0 to 1 h, from 1 to 6 h, from 3 to 12 h and from 12 to 24 h. The 6-h value was not si~~c~~y different from that for 12 h. In 35 S, the only non-significant difference among HOC values was registered between 3 and 6 h, HOC increasing strongly after all other exposure periods. Examining the effect of salinity at fixed time intervals showed that after the l-h exposure period, HOC was completeiy independent of external salinity. After the 3-h period, HOC remained ~ch~ged from 0 to 21 S but s~~c~t increases occurred from 14, 21 and 28 S to 35 S and from 0 to 28 S. After 6 h, HOC continued to be independent of salinity from 0 to 14 S but increased significantly from 14 to 2 1 and 35 S, and from 28 to 35 S. After the 12-h exposure, there were significant increases in HOC from 0 to 14,14 to 28 and 28 to 35 S while, after 24 h, HOC remained unchanged from 0 to 14 S, increasing si~~c~tly with each salinity increase above 14 S. It should be noted that mortality was only registered in very high salinities, occurring after 12-h exposure to 28 S and after 3 h in 3.5 S. Exposure time also afI’ects the point at which the hemolymph and the external medium attain osmotic ~u~ib~urn (Table I). Isosmotic points increase slowly during the fust 6 h but rise rapidly after the 12- and 24-h exposures. The data were also evaluated by means of a two-way analysis of variance to determine whether salinity, exposure time or their interaction afTect HOC. All factors significantly affected HOC (P c 0.0005).
The present study reveals that M. o~r~i~ demons~ates considerable ~e~b~ty of osmotic adjustment to salinity variation as a function of exposure time. Temporally the hemolymph osmotic response appears to consist of three phases. Firstly, there is an immediate increase in HOC during the first hour of exposure to all salinities (7 to 35 S), the magnitude of which appears to depend on the gradient between the internal and external osmotic concen~ations, i.e., HOC increases by some 35 mOsm (10%) in low salinities (5 14 S) and by about 85 mOsm (25 %) in higher salinities. Subsequently, in
TABLE
I
--_
1 3 6 12 24
380.6 + 12.9 380.1 + 13.8 346.0 c 20.6 334.1 + 22.5 313.6 rt: 12.9
-.
399.1 & 9.1 371.4 + 15.1 382.4 + 8.1 412.1 + 13.9 441.1 + 18.4
393.9 + 407.0 f 427.3 + 461.8 1 423.4 f
22.6 9.0 10.7 10.5 13.8
449.8 + 496.3 + 578.2 + 499.1 + 639.9 +
16.8 1.0 12.6 22.4 10.3
448.3 + 440.9 + 535.6 f 614.1 f 790.9 f
28 12.9 14.3 18.8 19.1 12.2
433.8 + 20.4 574.1 rf: 18.8 623.5 f 20.5 820.4 f 16.6 931.4 1 13.7
35
403.7 405.4 430.7 486.1 746.8
point
Hemolymph osmotic concentrations and isosmotic points in Mucrubruchium orfersii after acute exposure to various salinity-time combinations. Data are given as milh-osmoles per kilogram water and represent the mean k SEM (N = 11). -~ -.-,--_.. ~. -____ Salinity (S) Exposure Isosmotic 0 7 14 21 time (h)
&
%
; 5:
OSMOTIC REGULATION IN MACROBRQCHfUM
249
the second phase, there is a 6-h period of osmotic stability confined to the lower salinity exposures, i.e., 7 and 14 S. In higher salinities (2 21 S), there is a continuous increase with exposure time, attaining some 160% of the mean freshwater value. In the third phase, after the 6-h period, osmotic adjustment is less consistent in pattern. In 7 S, HOC does not appear to alter with exposure time; although HOC values increase continually, they do not rise more than 20% above that of freshwater-exposed shrimps. In 14 S, a distinct regulatory response is apparent after 12-h exposure when HOC is maximal, the 24-h HOC value not differing from that for 1 h. The clearest regulatory patterns are exhibited in 21 and 28 S. After a peak at 6 h, strong osmotic regulation of the hemolymph continues until 12 h after which HOC values increase substantially to 24 h (180 to 220% of freshwater HOC values). In sea water (35 S), regulatory ability is completely lost and HOC increases drastically, reaching some 250% of the freshwater HOC value after 24 h. Considering the salinity effect alone, at constant exposure intervals, two further tendencies are evident. In the l- to 3-h exposure groups, there is a stable, virtually salinity ~dependent response from 0 to 14 S, and another, less stable, from 21 to 28 S. There is also an inflexion point between 14 and 21 S which links the two more stable portions of the osmotic response. However, in the 12 and 24 h groups, only the stable, lower salinity portion persists as HOC increases markedly with salinity increase in the higher salinities. This phenomenon is clearly reflected in the values for isosmoticity which increase with exposure time (Table I). Thus, M. o&&ii exhibits several stages of osmotic behavior associated with the duration of exposure to external salinity, passing from a hyper-hypo-osmotic regulatory pattern after z l- to 6-h exposure through a stage of hyper-hypo-conformation after 12 h, finally becoming limited to hyperregulation after 24-h exposure. The data also indicate that in terms of osmotic adjustments and consequent survival ability, M. o&&i, although a freshwater shrimp, is still adequately equipped to confront acute changes in external salinity. This is achieved primarily by means of strong hyperregulation in low to midrange salinities (anisosmotic extracellular regulation; see Gilles (1979) for discussion). Some form of extracellular adjustment also evidently exists since HOC hypoconforms with external osmotic concentration in high salinities after some 6-h exposure. Tan 8z Choong (1981) have demonstrated the existence of intracellular osmotic regulation in iU. rosenbergii where, during hyperosmotic stress ( $t:30 S), hemolymph proteins are apparently hydrolyzed into free amino acids which are rapidly taken up by muscle tissues. This latter type of intracellular adjustment may occur naturahy in shrimp populations from the lower stream reaches which can be subjected to high salinities for a m~um of some 6 h during high tide. However, since permanent brackish water populations of M. olfrsi~ have not been encountered on the Szio Paul0 coast, it would appear that this shrimp has already made the position to freshwater, although, physiolo~c~y, it still pertains more to the brackish water crustacean fauna than to the older freshwater inhabitants like the atyid shrimps and crayfish, and the amphipod Gammarus pulex (see Shaw, 1961; Born, 1968 for discussion). This is
2.50
J. C. M~NAMA~A
pickily reflected in the high I-IOC seen in freshwater-expose ~~~~~~r~c~j~~ o&&xiiand in the lability of the isosmotic point which increases with increasing salinity, in addition to the dependence of the many larval stages on moderately saline water (17 to 21 S, McNamara ef al., 1986) for sustained larval development. In conclusion, it has been possible to interpret some aspects of osmo-ionic equilibrium in M. oIfrsii in terms of the independence of this species from the marine/estuarine environment and to evaluate the degree of adaptation to the freshwater biotope. In view of the characteristics outlined above, M. o&&i appears to be a recent, although efficient colonizer of the freshwater habitat.
The author wishes to express his gratitude to Dr. G. S. Moreira for the use of laboratory space and facilities during the course of this study, and to Dr. L. C. Salomao for helpful discussion. This research, conducted at the Centro de Biologia Marinha, Universidade de S%o Paulo, in Slxo Sebastiao, was financed by research grants (81/1855-7; 81/1854-O; 83/0788-O) from the FundaG?lo de Amparo ii Pesquisa do Estado de S&o Paulo.
REFERENCES ARMSTRONG,D.A., K. STRANGE,3. CROWE,A. KNIGHT & M. SIMMONS,1981. High salinity acclimation by the prawn Macrobrachium wsenbergti: uptake of exogenous ammonia and changes in endogenous nitrogen compounds, Biol. Bull. (Woods Hole, Mass.), Vol. 160, pp. 349-365. BEADLE,L.C., 1943. Osmotic regulation and the faunas of inland waters. Biol. Rev., Vol. 18, pp. 172-183. BORN, J. W., 1968. Osmoregulatory capacities of two c&dean shrimps, Syncarts pacifica (Atyidae) and Palaemon macrodactylw (Palaemonidae). Biol. Bull. (Woods Hole, Mass.), Vol. 134, pp. 235-244. CASTILLE,F.L. & A.L. LAWRENCE,1981. The effect of salinity on the osmotic, sodium and chloride concentrations in the hemolymph of the freshwater shrimps Macrobrachium ohione Smith and Macrobrachium rosenbergii De Man. Comp. Biochem. Physioi.., Vol. 70A, pp. 47-50. GILLES, R., 1979. Intracellular osmotic effecters. In, Mechanisms ofosmoregulation in animals, edited by R. Gilles, John Wiley & Sons, Chichester, pp. 111-154. HEDGP~X, J. W., 1949. The North American species of Ma~obruch~. Texas .I. Sci., Vol. 1, pp. 28-38. HEDGPETH,J. W., 1957. Estuaries and lagoons. II. Biological aspects. In, Treatise on marine ecology and pa~ae~co~, edited by J.W. Hedgpeth, &of. Sot. Am., Mem. 67, Vol. 1, pp. 693-729. KIRKPATRICK,K. & M. B. JONES, 1985. Salinity tolerance and osmoreguiation of a prawn, Palaemon a&&Milne Edwards (Caridea, Palaemonidae). J. Exp. Mar. Biol. Bcol., Vol. 93, pp. 61-70. KNOWLTON,R.E. & D. F. KIRBY, 1984. Salinity tolerance and sodium balance in the prawn Palaemonefes pugio Holthuis, in relation to other Palaemonetes spp. Comp. Btochem. Physiol., Vol. 17A, pp. 425-430. MCNAMARA,J. C., G. S. MOREIRA& SC. SOUZA, 1986. The effect of salinity on respiratory metabolism in selected ontogenetic stages oftbe freshwater shrimp Macrobrachium orfrsii (Decapoda, Palaemonidae). Camp. Biochem. Physiol., Vol. 83A, pp. 359-364. MOREIRA, G.S., J.C. MCNAMARA, S.E. SSUMWAY & P.S. MOREIRA, 1983. -0smoregulation and respiratory metabolism in Brazilian Macrobrachium (Decapoda, Palaemonidae). Comp. Biochem. Physiol., Vol. 74A, pp. 57-62. ORTMANN,A. E., 1902. The geographical distribution of freshwater decapods and its bearing upon ancient geography. Proc. Am. Phil. Sot., Vol. 41, pp. 267-400.
OSMOTIC REGULATION IN MACROBRACHIUM
251
READ, G.H., 1984. Intraspecific variation in osmoregulatory capacity of larval, post larval, juvenile and adult Macrobrachium petersi (Hilgendorf). Comp. Biochem. Physiol., Vol. 78A, pp. 501-506. SHAW,J., 1961. Sodium balance in Eriocheir sinensis (M. Edw.). The adaptation of the Crustacea to fresh water. J. Exp. Biol., Vol. 38, pp, 153-162. TAN, C. H. & K. Y. CHOONG, 1981. Effect of hyperosmotic stress on hemolymph protein, muscle ninhydrinpositive substances and free amino acids in Macrobrachium rosenbergii (De Man). Comp. Biochem. Physiol., Vol. 70A, pp. 485-489. ZAR, J.H., 1974. Biostatistical analysis. Prentice-Hall, Englewood Cliffs, 620 pp.
.I. Exp. Mar. BioL Ecol., 1987, Vol. 107, pp. 245-251 Eisevier
JEM 00858
The time course of osmotic regulation in the freshwater shrimp Macrobrachium olfrsii (Wiegmann) (Decapoda, Palaemonidae) John C. McNamara ~e~~rtarne~to de ~~~o~o~.a,~~ti~to de Bioci&cias, U~iveTsidade de &To Pa&,
&lo Pa&o. Brazil
(Received 10 September 1986; revision received 8 January 1987; accepted 15 January 1987) Abstract: Alterations in hemolymph osmotic concentration (HOC) in the decapod shrimp Macrobrachium olfersii (Wiegmann) were examined as a function of salinity (0, 7, 14,21,28 and 35 S) and exposure time (1,3,6,12 and 24 h). This freshwater palaemonid maintains a high HOC in river water (362.9 + 7.8 mOsm) which is virtually unaffected by salinity variation up to 14 S or exposure time. However, in 21 and 28 S, HOC increases rapidly until 6 h, remaining stable to 12 h, then increasing to 24 h; in 35 S, HOC increases continually with time. M. olfersii hyperregnlates its HOC in salinities up to isosmotic (14 S), exhibiting various degrees of HOC response (strong hyporegnlation to isosmoticity) in higher salinities, as a function of time. Hemoiymph isosmotic points increase little during the first 6 h of exposure, rising rapidly after 12 and 24 h. Given its high HOC in freshwater, tolerance of high salinities and the dependence of its larval stages on saline water, M. o&r&is considered to be a recent, ~thou~ efficient, colonizer of the freshwater biotope. Key words: Macrobrachium; Osmotic regulation; Freshwater shrimp; Salinity exposure
Freshwater shrimps, particularly those of the genus Macrobrachium, are apparently still in the process of penetrating the freshwater biotope (Ortmann, 1902; Hedgpeth, 1949, 1957). Such decapod crustaceans exhibit numerous biological characteristics indicative of the recency of this invasion, e.g., high hemol~ph osmo-ionic concentrations, tolerance of high salinities, marked dependence on saline water for larval development with many larval stages, and migratory behavior. Evidently, the development of an efficient osmoregulatory capabiity is of fundamental importance to the colonization of the freshwater habitat by such shrimps since these maintain high ionic concentrations in the body fluids against a strong gradient, implying considerable energetic expense (Moreira et al., 1983). Investigation of osmo-ionic equilibrium in the hemolymph permits evaluation of the establishment, maintenance and efficiency of the osmotic response which, together with the biological characteristics of the species, should provide a measure of its capability to invade the freshwater biotope Correspondence address: John C. McNamara, Departamento de Fisiologia, Instituto de Cikrcias Biomedicas, CP 4365, Universidade de Sfio Paulo, OlOOOS&J Paulo, Brazil. 0022-~81/87/$03.50 0 1987 Elsevier Science Pnbhshers B.V. (Biomedic~ Division)
246
J. C. McNAMARA
and the adaptive mechanisms concerned (see Beadle, 1943; Shaw, 1961). It is also interesting to evaluate the extent to which other major energy consuming processes such as respiratory meta~lism and excretion, for example, are affected by hemol~ph osmotic response. Some aspects of both osmoregulatory phenomena (see Armstrong et al., 198 1; Castille & Lawrence, 198 1; Moreira et al., 1983; Read, 1984 for references) and respiratory metabolism (see Moreira et&, 1983; McNamara et al., 1986 for references) have been examined in Macrobracbium, and in considerable detail in other brackish water and marine palaemo~d shrimps (see Knowlton & Kirby, 1984; Kirkpatrick & Jones, 1985 for references). Relatively little is known about the regulation of hemolymph osmotic concentration in freshwater shrimps (see Moreira et al., 1983; Read, 1984) and information is especially lacking on the establishment of osmotic response patterns to acute sahnity exposure. Thus, in the present study, I have investigate the time course of hemol~ph osmotic response to salinity variation in a small palaemonid shrimp, M. oifersii (Wiegmarm), in an attempt to evaluate the degree of osmotic adaptation to freshwater in this decapod typical of the coastal fluvial system of southern Brazil. MATERIALS AND METHODS
Specimens of the freshwater shrimp M. o&xv?, 5 to 7 cm total length, were collected from the marginal vegetation of the GuaecL River (23 ‘49’S : 45’ 27’W) on the northern coast of the State of Sao Paulo, Brazil. In the laboratory, the shrimps were rn~nt~~ unfed, in plastic tanks containing 15 1 of aerated river water, at room temperature (x 25 ‘C), for 1 or 2 days prior to experimentation. To evaluate the effect of salinity and exposure time on hemolymph osmotic responses, groups of 15 to 20 intermolt shrimps were exposed for 1, 3,6, 12 or 24 h to media of 0, 7, 14, 21, 28 or 35 S* in a constant temperature chamber fmished with a 12-h light : 12-h dark photoperiod, at 20 “C. All salinities were prepared from Guaecit River water (< 1 S) and Sao Sebastigo Channel sea water (35 S), To obtain hemolymph samples, after each exposure period, the pericardial region of each shrimp was punctured with a Number 25-8 needle and 70 to 100 ~1 of lluid drawn into a 1 ml plastic syringe. The entire volume was then transferred to a 1.5 ml Teflon flask and a 50 ~1 subsample taken with an automatic pipette. The osmotic concentration of this subsample was immediately determined using a Knauer semi-micro osmometer. All results are presented as mOsm/kg water (mean _+SEM).The data were evaluated using the Student-Neck-Keuls procedure and a two-way analysis of variance according to Zar (1974).
* Practicalsalinity:definition (UNESCO TechnicaI Papers in Marine Science, Number 39, Vol. 3, p. 11).
OSMOTICREGULATIONIN ~ACROB~CHIU~
247
RESULTS Data demons~ating the effect of exposure time and salinity on the hemol~ph osmotic ~oncen~ation (HOC) ofM. orfrsii are presented in Table I. In river water (0 S), the normal external medium, HOC remained stable at 362.9 4 7.8 mOsm/kg (n = 55) (mean of all river water exposure times) and was independent of exposure time. In 7 S, no significant differences were recorded among the diBerent exposure times although there was a tendency for HOC to increase with time. In 14 S, the only si~c~t increase was recorded between 0- and 12-h exposure. In 21 S, HOC increased signiticantly from 0 to 1 h and again from 1 to 6 h. There was no difference between the values for 6 and 12 h; HOC increased again significantly from 12to 24 h. A very similar pattern was observed in 28 S, HOC increasing significantly from 0 to 1 h, from 1 to 6 h, from 3 to 12 h and from 12 to 24 h. The 6-h value was not si~~c~~y different from that for 12 h. In 35 S, the only non-significant difference among HOC values was registered between 3 and 6 h, HOC increasing strongly after all other exposure periods. Examining the effect of salinity at fixed time intervals showed that after the l-h exposure period, HOC was completeiy independent of external salinity. After the 3-h period, HOC remained ~ch~ged from 0 to 21 S but s~~c~t increases occurred from 14, 21 and 28 S to 35 S and from 0 to 28 S. After 6 h, HOC continued to be independent of salinity from 0 to 14 S but increased significantly from 14 to 2 1 and 35 S, and from 28 to 35 S. After the 12-h exposure, there were significant increases in HOC from 0 to 14,14 to 28 and 28 to 35 S while, after 24 h, HOC remained unchanged from 0 to 14 S, increasing si~~c~tly with each salinity increase above 14 S. It should be noted that mortality was only registered in very high salinities, occurring after 12-h exposure to 28 S and after 3 h in 3.5 S. Exposure time also afI’ects the point at which the hemolymph and the external medium attain osmotic ~u~ib~urn (Table I). Isosmotic points increase slowly during the fust 6 h but rise rapidly after the 12- and 24-h exposures. The data were also evaluated by means of a two-way analysis of variance to determine whether salinity, exposure time or their interaction afTect HOC. All factors significantly affected HOC (P c 0.0005).
The present study reveals that M. o~r~i~ demons~ates considerable ~e~b~ty of osmotic adjustment to salinity variation as a function of exposure time. Temporally the hemolymph osmotic response appears to consist of three phases. Firstly, there is an immediate increase in HOC during the first hour of exposure to all salinities (7 to 35 S), the magnitude of which appears to depend on the gradient between the internal and external osmotic concen~ations, i.e., HOC increases by some 35 mOsm (10%) in low salinities (5 14 S) and by about 85 mOsm (25 %) in higher salinities. Subsequently, in
TABLE
I
--_
1 3 6 12 24
380.6 + 12.9 380.1 + 13.8 346.0 c 20.6 334.1 + 22.5 313.6 rt: 12.9
-.
399.1 & 9.1 371.4 + 15.1 382.4 + 8.1 412.1 + 13.9 441.1 + 18.4
393.9 + 407.0 f 427.3 + 461.8 1 423.4 f
22.6 9.0 10.7 10.5 13.8
449.8 + 496.3 + 578.2 + 499.1 + 639.9 +
16.8 1.0 12.6 22.4 10.3
448.3 + 440.9 + 535.6 f 614.1 f 790.9 f
28 12.9 14.3 18.8 19.1 12.2
433.8 + 20.4 574.1 rf: 18.8 623.5 f 20.5 820.4 f 16.6 931.4 1 13.7
35
403.7 405.4 430.7 486.1 746.8
point
Hemolymph osmotic concentrations and isosmotic points in Mucrubruchium orfersii after acute exposure to various salinity-time combinations. Data are given as milh-osmoles per kilogram water and represent the mean k SEM (N = 11). -~ -.-,--_.. ~. -____ Salinity (S) Exposure Isosmotic 0 7 14 21 time (h)
&
%
; 5:
OSMOTIC REGULATION IN MACROBRQCHfUM
249
the second phase, there is a 6-h period of osmotic stability confined to the lower salinity exposures, i.e., 7 and 14 S. In higher salinities (2 21 S), there is a continuous increase with exposure time, attaining some 160% of the mean freshwater value. In the third phase, after the 6-h period, osmotic adjustment is less consistent in pattern. In 7 S, HOC does not appear to alter with exposure time; although HOC values increase continually, they do not rise more than 20% above that of freshwater-exposed shrimps. In 14 S, a distinct regulatory response is apparent after 12-h exposure when HOC is maximal, the 24-h HOC value not differing from that for 1 h. The clearest regulatory patterns are exhibited in 21 and 28 S. After a peak at 6 h, strong osmotic regulation of the hemolymph continues until 12 h after which HOC values increase substantially to 24 h (180 to 220% of freshwater HOC values). In sea water (35 S), regulatory ability is completely lost and HOC increases drastically, reaching some 250% of the freshwater HOC value after 24 h. Considering the salinity effect alone, at constant exposure intervals, two further tendencies are evident. In the l- to 3-h exposure groups, there is a stable, virtually salinity ~dependent response from 0 to 14 S, and another, less stable, from 21 to 28 S. There is also an inflexion point between 14 and 21 S which links the two more stable portions of the osmotic response. However, in the 12 and 24 h groups, only the stable, lower salinity portion persists as HOC increases markedly with salinity increase in the higher salinities. This phenomenon is clearly reflected in the values for isosmoticity which increase with exposure time (Table I). Thus, M. o&&ii exhibits several stages of osmotic behavior associated with the duration of exposure to external salinity, passing from a hyper-hypo-osmotic regulatory pattern after z l- to 6-h exposure through a stage of hyper-hypo-conformation after 12 h, finally becoming limited to hyperregulation after 24-h exposure. The data also indicate that in terms of osmotic adjustments and consequent survival ability, M. o&&i, although a freshwater shrimp, is still adequately equipped to confront acute changes in external salinity. This is achieved primarily by means of strong hyperregulation in low to midrange salinities (anisosmotic extracellular regulation; see Gilles (1979) for discussion). Some form of extracellular adjustment also evidently exists since HOC hypoconforms with external osmotic concentration in high salinities after some 6-h exposure. Tan 8z Choong (1981) have demonstrated the existence of intracellular osmotic regulation in iU. rosenbergii where, during hyperosmotic stress ( $t:30 S), hemolymph proteins are apparently hydrolyzed into free amino acids which are rapidly taken up by muscle tissues. This latter type of intracellular adjustment may occur naturahy in shrimp populations from the lower stream reaches which can be subjected to high salinities for a m~um of some 6 h during high tide. However, since permanent brackish water populations of M. olfrsi~ have not been encountered on the Szio Paul0 coast, it would appear that this shrimp has already made the position to freshwater, although, physiolo~c~y, it still pertains more to the brackish water crustacean fauna than to the older freshwater inhabitants like the atyid shrimps and crayfish, and the amphipod Gammarus pulex (see Shaw, 1961; Born, 1968 for discussion). This is
2.50
J. C. M~NAMA~A
pickily reflected in the high I-IOC seen in freshwater-expose ~~~~~~r~c~j~~ o&&xiiand in the lability of the isosmotic point which increases with increasing salinity, in addition to the dependence of the many larval stages on moderately saline water (17 to 21 S, McNamara ef al., 1986) for sustained larval development. In conclusion, it has been possible to interpret some aspects of osmo-ionic equilibrium in M. oIfrsii in terms of the independence of this species from the marine/estuarine environment and to evaluate the degree of adaptation to the freshwater biotope. In view of the characteristics outlined above, M. o&&i appears to be a recent, although efficient colonizer of the freshwater habitat.
The author wishes to express his gratitude to Dr. G. S. Moreira for the use of laboratory space and facilities during the course of this study, and to Dr. L. C. Salomao for helpful discussion. This research, conducted at the Centro de Biologia Marinha, Universidade de S%o Paulo, in Slxo Sebastiao, was financed by research grants (81/1855-7; 81/1854-O; 83/0788-O) from the FundaG?lo de Amparo ii Pesquisa do Estado de S&o Paulo.
REFERENCES ARMSTRONG,D.A., K. STRANGE,3. CROWE,A. KNIGHT & M. SIMMONS,1981. High salinity acclimation by the prawn Macrobrachium wsenbergti: uptake of exogenous ammonia and changes in endogenous nitrogen compounds, Biol. Bull. (Woods Hole, Mass.), Vol. 160, pp. 349-365. BEADLE,L.C., 1943. Osmotic regulation and the faunas of inland waters. Biol. Rev., Vol. 18, pp. 172-183. BORN, J. W., 1968. Osmoregulatory capacities of two c&dean shrimps, Syncarts pacifica (Atyidae) and Palaemon macrodactylw (Palaemonidae). Biol. Bull. (Woods Hole, Mass.), Vol. 134, pp. 235-244. CASTILLE,F.L. & A.L. LAWRENCE,1981. The effect of salinity on the osmotic, sodium and chloride concentrations in the hemolymph of the freshwater shrimps Macrobrachium ohione Smith and Macrobrachium rosenbergii De Man. Comp. Biochem. Physioi.., Vol. 70A, pp. 47-50. GILLES, R., 1979. Intracellular osmotic effecters. In, Mechanisms ofosmoregulation in animals, edited by R. Gilles, John Wiley & Sons, Chichester, pp. 111-154. HEDGP~X, J. W., 1949. The North American species of Ma~obruch~. Texas .I. Sci., Vol. 1, pp. 28-38. HEDGPETH,J. W., 1957. Estuaries and lagoons. II. Biological aspects. In, Treatise on marine ecology and pa~ae~co~, edited by J.W. Hedgpeth, &of. Sot. Am., Mem. 67, Vol. 1, pp. 693-729. KIRKPATRICK,K. & M. B. JONES, 1985. Salinity tolerance and osmoreguiation of a prawn, Palaemon a&&Milne Edwards (Caridea, Palaemonidae). J. Exp. Mar. Biol. Bcol., Vol. 93, pp. 61-70. KNOWLTON,R.E. & D. F. KIRBY, 1984. Salinity tolerance and sodium balance in the prawn Palaemonefes pugio Holthuis, in relation to other Palaemonetes spp. Comp. Btochem. Physiol., Vol. 17A, pp. 425-430. MCNAMARA,J. C., G. S. MOREIRA& SC. SOUZA, 1986. The effect of salinity on respiratory metabolism in selected ontogenetic stages oftbe freshwater shrimp Macrobrachium orfrsii (Decapoda, Palaemonidae). Camp. Biochem. Physiol., Vol. 83A, pp. 359-364. MOREIRA, G.S., J.C. MCNAMARA, S.E. SSUMWAY & P.S. MOREIRA, 1983. -0smoregulation and respiratory metabolism in Brazilian Macrobrachium (Decapoda, Palaemonidae). Comp. Biochem. Physiol., Vol. 74A, pp. 57-62. ORTMANN,A. E., 1902. The geographical distribution of freshwater decapods and its bearing upon ancient geography. Proc. Am. Phil. Sot., Vol. 41, pp. 267-400.
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READ, G.H., 1984. Intraspecific variation in osmoregulatory capacity of larval, post larval, juvenile and adult Macrobrachium petersi (Hilgendorf). Comp. Biochem. Physiol., Vol. 78A, pp. 501-506. SHAW,J., 1961. Sodium balance in Eriocheir sinensis (M. Edw.). The adaptation of the Crustacea to fresh water. J. Exp. Biol., Vol. 38, pp, 153-162. TAN, C. H. & K. Y. CHOONG, 1981. Effect of hyperosmotic stress on hemolymph protein, muscle ninhydrinpositive substances and free amino acids in Macrobrachium rosenbergii (De Man). Comp. Biochem. Physiol., Vol. 70A, pp. 485-489. ZAR, J.H., 1974. Biostatistical analysis. Prentice-Hall, Englewood Cliffs, 620 pp.